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PODIUM PRESENTATIONS Objective Recent studies have suggested that the subchondral bone P1 metabolism is intimately involved in cartilage degradation in osteoarthritis Autoantibodies to Heat Shock Protein 60 (hsp60) Promote Thrombus (OA). Bone remodelling is tightly regulated by a molecular triad: Formation in a Murine Model of Arterial Thrombosis OPG/RANK/RANKL. RANKL enhances osteoclastogenesis via interaction Melanie Dieude (Division of Rheumatology, The Research Institute of the with its receptor RANK, whereas OPG inhibits this osteoclastogenesis by act- McGill University Health Centre), Marc-Antoine Gillis (The Montreal Heart ing as a soluble RANKL receptor competitor. Depending on the low or high Institute, University of Montreal, Montreal), Jean-Francois Theoret (The level of PGE2, human OA subchondral osteoblasts can be discriminated into Montreal Heart Institute, University of Montreal, Montreal), Jerrold S. Levine two subpopulations: low (L) or high (H), respectively. (Section of Nephrology, University of Illinois at Chicago, Chicago, USA), Methods We investigated the modulation of OPG and RANKL levels and Eric Thorin (The Montreal Heart Institute, University of Montreal, Montreal), RANKL1 and 3 isoforms by real-time PCR, and osteoclast formation using Yahye Merhi (The Montreal Heart Institute, University of Montreal, the pre-osteoclastic cell line RAW 264.7, comparing the effects of normal to Montreal), Joyce Rauch (Division of Rheumatology, The Research Institute each OA osteoblast subpopulation (L or H). Human knee subchondral bone of the McGill University Health Centre) was also examined histologically. Further, we determined by flow cytometry Anti-heat shock protein 60 (anti-hsp60) autoantibodies from systemic lupus on L and H OA osteoblasts the level and modulation of membranous RANKL erythematosus patients induce endothelial cell apoptosis in vitro, and are upon treatment with/without osteotropic factors. associated with an increased frequency of thrombosis in patients with lupus Results Histological analysis revealed that the L OA osteoblasts had reduced anticoagulant antibodies (Dieudé et al. Arthritis Rheum 2004). However, the subchondral bone mass compared to normal, in contrast to the H OA which mechanisms responsible for anti-hsp60 autoantibody induction and pathogen- showed an increase. The OPG/RANKL mRNA ratio was significantly esis remain unclear. decreased in L OA compared to normal or H OA, and markedly increased in Objective We aimed to study how autoantibodies to murine hsp60 could be H OA compared to normal. This was further supported by the membranous induced in nonautoimmune mice, and to determine if these autoantibodies RANKL levels which were significantly elevated in L OA compared to the H would promote thrombus formation in an in vivo model of carotid artery OA and normal cells. Moreover, L OA but not H OA cells induced a signifi- thrombosis. cantly higher level of osteoclast differentiation and formation. Experiments Methods BALC/c mice were immunized with bacterial hsp65 and Freund’s with osteotropic factors revealed that i) in L OA osteoblasts, TNF-alpha, IL- adjuvant, or adjuvant alone, to produce IgG antibodies reactive with murine 1beta, PGE2, IL-6 and Vitamin D3 significantly increased the membranous hsp60 or control antibodies respectively. Naïve BALB/c mice were treated RANKL level, whereas ii) in H OA none of the factors tested affected with either purified polyclonal anti-murine hsp60 IgG antibodies or control RANKL. Finally, RANKL isoforms 1 and 3 were significantly higher in the IgG 48 hours prior to inducing carotid artery injury with FeCl3. On the day L OA compared to H OA. of the experiment, mice were anesthetized and the right carotid artery was Conclusion The OPG/RANKL ratio in the two human OA subchondral bone exposed. Blood flow through the carotid artery was measured using a minia- osteoblast subpopulations indicates different stages of this diseased tissue, in ture ultrasound flow probe interfaced with a flow meter and a computer-based which the L OA osteoblasts favour bone resorption. A better understanding of data acquisition program. The blood flow was monitored pre-FeCl3-induced the modulation of subchondral bone RANKL during the OA process will be injury to establish normal flow, and post-injury to follow thrombus formation. useful in the development of a specific therapeutic target. Blood flow decreased when thrombus formation occurred and stopped when the thrombus completely occluded the carotid artery. Blood was collected by P3 cardiac puncture for whole blood platelet aggregation studies. Elevated BAFF Levels Correlate with Altered B Cell Phenotypes in Results All (n=14) mice treated with anti-hsp60 antibodies developed virtual- Patients with Systemic Lupus Erythematosus ly complete occlusion of the carotid artery, with a mean blood flow of 1.86% Carolina Landolt-Marticorena (Toronto Western Research Institute), Gabriel following thrombus formation: no reperfusion was observed. In contrast, mice Bonventi (Toronto Western Research Institute), Yongchun Cai (Toronto (n=14) treated with control IgG showed greater instability of the thrombus Western Research Institute), Thulasi Unnithan (Toronto Western Research with frequent reperfusion [7/14 mice]) and a mean blood flow of 22.3% fol- Institute), Dafna Gladman (Toronto Western Research Institute), Murray lowing thrombus formation. In vitro collagen-induced platelet aggregation Urowitz (Toronto Western Research Institute), Paul R. Fortin (Toronto using blood from these mice was significantly lower in anti-hsp60-treated than Western Research Institute), Joan E. Wither (Toronto Western Research in control IgG-treated mice (mean resistance: 6.71 versus 10.38 Ohms, p = Institute) 0.017), consistent with prior in vivo activation in the anti-hsp60 mice. Objective It has been demonstrated that lupus patients have elevated serum Conclusion Our findings demonstrate that: i) anti-hsp60 autoantibodies can and RNA levels of BAFF. In transgenic mouse models, BAFF has ubiquitous be induced in nonautoimmune individuals by exposure to a bacterial antigen; effects including rescue of auto-reactive transitional B cells, enhancement of and ii) in a murine model of arterial thrombosis, passively transferred anti- T-B cell collaboration, and expansion of plasma cells. Since these changes hsp60 autoantibodies promote thrombus formation, vascular occlusion, and are similar to those observed in lupus patients, we examined whether BAFF thrombus stabilization. These data provide support for a prothrombotic role of expression correlates with alterations in the peripheral blood B cell compart- anti-hsp60 autoantibodies. Work supported by CIHR and FRSQ “Réseau en ments of lupus patients. santé cardiovasculaire”. Methods Patients (n = 58) satisfying ≥ 4 ACR criteria were recruited from the University of Toronto Lupus Clinic. Healthy controls (n =11) P2 were also recruited. Clinical and demographic information was obtained Human OA Subchondral Bone Remodelling Is Closely Dependent on for all study participants. Disease activity was measured by the RANKL Modulation SLEDAI-2K, with a score of ≥ 7 defined as high disease activity. Steeve Kwan Tat (Osteoarthritis Research Unit, University of Montreal Peripheral blood mononuclear cells from all study participants were iso- Hospital Centre, Notre-Dame Hospital), Jean-Pierre Pelletier (Osteoarthritis lated, stained with various combinations of fluorescently labeled anti- Research Unit, University of Montreal Hospital Centre, Notre-Dame bodies, and analyzed by flow cytometry. RNA was isolated from periph- Hospital), Daniel Lajeunesse (Osteoarthritis Research Unit, University of eral blood and converted to cDNA using a ABI high capacity kit. BAFF Montreal Hospital Centre, Notre-Dame Hospital), Hassan Fahmi RNA expression was measured by qRT-PCR reaction using Taqman Low (Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre- Density Arrays and normalized to a housekeeping gene (GAPDH). Dame Hospital), Nicolas Duval (Centre de Convalescence Pavillon des Statistical differences between groups where determined by the Mann- Charmilles), Johanne Martel-Pelletier (Osteoarthritis Research Unit, Whitney test. University of Montreal Hospital Centre, Notre-Dame Hospital) Results BAFF RNA expression was significantly elevated in SLE patients as Personal non-commercial use only. The Journal of Rheumatology Copyright © 2008. All rights reserved. CRA - Podium presentations 1171 Downloaded on September 27, 2021 from www.jrheum.org compared to controls (p < 0.0001) and showed an association with high dis- Health Center), Patrick Bélisle (McGill University Health Center), Ann E. ease activity (p = 0.017). As we, and others have shown, lupus patients had Clarke (McGill University Health Center), Christian A. Pineau (McGill significantly elevated levels of pre-germinal center (p < 0.0001) and plasma University Health Center) cells (p = 0.0352) as compared to controls. In the current and previous stud- Objective Over the past three decades, in North America, the proportion of ies, we have noted a trend towards an expansion of the transitional cell com- women training in rheumatology has increased considerably, but there is con- partment in lupus patients when compared to controls. Patients were stratified cern that their academic productivity is inferior to their male counterparts. into high and low levels for each B cell population based on whether the pro- Because publication in medical journals

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